PROJECT SUMMARY The primary focus in the proposed R33 phase is to advance the biological technology for large-scale manufacturing of the T-cell Biofactory and to apply it to mitigate drug resistance and immunosuppression in the context of targeting solid tumors. The T-cell Biofactory is responsive to the IMAT's R33 mechanism because it is an in vivo vector platform technology for synthesizing engineered proteins at the tumor site and in proportion to the disease burden. The tumor microenvironment (TME) has been implicated in drug resistance and immunosuppression that are major contributors to the failure of current approaches in cancer therapies. The TME is supported within the dense fibrous extracellular matrix (ECM) tissue growth that is common in many solid tumors (prostate, colon, breast, stomach, pancreatic, and ovarian) and its systemic targeting causes side effects that promote cachexia and morbidity. Hence, the challenge is to selectively break down the ECM?in which the TME resides?without affecting normal tissues. Investigators' long-term goal is to develop new treatments for cancer by harnessing the potential of the immune system to target underlying mechanisms in the tumor pathology. The objective in the R33 phase, toward this long-term goal, is to validate the T-cell Biofactory in preclinical trials to selectively break down the ECM in a mouse model of epithelial ovarian cancer (EOC). To achieve this objective, the investigators' strategy is to engineer the T-cell Biofactory to synthesize a protease for degrading ECM upon engaging the Folate Receptor alpha (FRa) antigens overexpressed on ovarian cancer cells. As such, the T-cell Biofactory can express calibrated amounts of any human or non- human protein with desired properties directly at the disease site, thereby sparing healthy cells from damaging side effects. The rationale for this effort is that it will lead to a technology that can be scaled up for Phase I/II clinical trials, to locally disrupt the ECM and reset the TME for a positive treatment outcome. The investigators have selected EOC to represent other solid tumors but the T-cell Biofactory is a technology-driven platform that will be reprogrammable to target other solid tumors. The investigations for developing the technology will be conducted by: (Aim 1) engineering the T-cell Biofactory to autonomously express protease upon engaging the FRa antigen on the EOC cells; and (Aim 2) introducing biomimetic microbeads to rapidly expand the T-cell Biofactories. This project is significant because it will demonstrate the T-cell Biofactory technology for (1) controlled synthesis of proteases in the TME without affecting healthy cells; (2) neutralization of drug resistance and immunosuppression in solid tumors; (3) cGMP-compliant scalable processes amenable to large-scale manufacturing; and (4) readiness for preclinical assessment. The innovation in this project is in utilizing the existing knowledge of the surface of the antigen-presenting cells for large-scale culturing of engineered T cells and in using the T-cell Biofactory to circumvent systemic effect of directly infused proteases.